Complement factor H-derived short consensus repeat 18-20 enhanced complement-dependent cytotoxicity of ofatumumab on chronic lymphocytic leukemia cells

Abstract

The antitumor activity of monoclonal antibodies in the treatment of chronic lymphocytic leukemia is mediated mainly by antibody-dependent cellular cytotoxicity and complement-dependent cytotoxicity. Unfortunately, the efficacy of complement-dependent cytotoxicity is strongly restricted due to the expression and acquisition of regulators of complement activation by lymphocytic leukemia cells. Whereas the role of membrane regulators of complement activation, such as CD55 and CD59, has been investigated in detail in chronic lymphocytic leukemia, the involvement of soluble regulators of complement activation, such as complement factor H, has not yet been reported. Propidium iodide staining was performed to investigate the efficacy of ofatumumab and factor H-derived shortconsensus-repeat 18-20 in the induction of complement-dependent cytotoxicity on primary chronic lymphocytic leukemia cells from 20 patients. Deposition of complement C3 fragments was monitored by western blot analysis. Expression of CD20, CD55 or CD59 was determined by FACS analysis. Replacement of factor H with short consensus repeat 18-20 significantly increased the susceptibility of primary chronic lymphocytic leukemia cells to ofatumumab-induced complement-dependent cytotoxicity. More importantly, addition of short-consensus-repeat 18-20 was able to overcome complement-resistance occurring during treatment with ofatumumab alone. Use of short consensus repeat 18-20 is likely to prolong the turnover time of active C3b fragments generated on the target cells following ofatumumab-induced complement activation, thereby improving specific killing of chronic lymphocytic leukemia cells by complement-dependent cytotoxicity. The relative contribution of factor H to the protection of chronic lymphocytic leukemia cells against complement-dependent cytotoxicity was comparable to that of CD55. Our data suggest that, by abrogating factor H function, short consensus repeat 18-20 may provide a novel approach that improves the complement-dependent efficacy of therapeutic monoclonal antibodies.

Regulation of complement and modulation of its activity in monoclonal antibody therapy of cancer

“…26 It was recently shown that the abrogation of fH function by using short-consensus repeat 18-20, representing the C-terminal ligand binding domain, in combination with ofatumumab or rituximab resulted in an increased susceptibility of primary CLL cells to CDC. 91,92 A further synergistic effect was seen upon blockage of fH and CD55/CD59. The decay of C3b to iC3b is strongly mediated by Factor I, for which most of the described CRPs exhibit cofactor function.…”

CD20 mAb-Mediated Complement Dependent Cytotoxicity of Tumor Cells is Enhanced by Blocking the Action of Factor I

“…The key "zero time" control in step 1 provides additional validation for the paradigm, because the cells in this sample (no deposited C3b) are not lysed when they are reacted in NHS-EDTA. Moreover, prolonged incubation of mAb-opsonized cells in step 1 should lead to Factor I-mediated decay of activated C3b to iC3b and then to C3d [45,46,[51][52][53][54], and the results illustrated in Figure 1A reveal that after a 30 min incubation in step 1, lysis of the cells in step 2 is reduced considerably, thus indicating that a substantial fraction of the deposited C3b fragments must have been degraded to inactive iC3b or C3d.…”

“…However, as noted above, prototype in vitro experiments have in fact demonstrated that inhibition of cell-associated complement control proteins CD55 and CD59, as well as inhibition of the action of soluble Factor H can all enhance CDC mediated by both of these mAbs [20,33,53,54,61], thus indicating there is indeed room for improvement. Moreover, levels of CDC higher than 90% are difficult to achieve with RTX or with OFA, even with cell lines, and this is particular evident when a clinically important target, i.e., primary CLL cells are examined [16,63].…”

“…Indeed, we now provide evidence that C3b deposition on a mAb-targeted cell can reach a saturating level, and that if all of this C3b is decayed to iC3b, then it will not be possible to kill this cell with complement, even if the mAb is still bound to the cells (Figures 1-3). Alternatively, both soluble factors as well as the complement control proteins expressed on the tumor cells, in common with normal cells, may limit mAb-mediated killing [42,43,53,54]. Therefore, focused strategies that can mitigate or limit exhaustion, or that can block the action of these factors have the potential to increase substantially killing of tumors by mAbs already approved by the FDA [20,22,30,33,53,54,61].…”

Monoclonal Antibodies in Cancer Therapy: Mechanisms, Successes and Limitations

“…C immune mediated tumour cell killing by engaging antibody-dependent-cell-mediated-cytotoxicity, complement-mediated-cytotoxicity and activating cellular phagocytosis (4,17,18). Additionally, immunostimulatory CmAbs can activate T lymphocyte cells through the inhibition of T lymphocyte inhibitory receptors (19).…”